Liquid enzyme preparations

ABSTRACT

Liquid enzyme preparations comprising at least one anhydrous organic liquid as a vehicle for one or more enzymes and methods for using such preparations e.g. in beamhouse operations in the commercial production of leather.

This application is a division of application No. 07/152,020 filed Feb.3, 1988.

The present invention relates to liquid enzyme preparations comprisingan anhydrous organic liquid as a vehicle for an enzyme, suitablytogether with an additive modifying the rheology of the composition, andto methods for making leather employing such preparations.

THE PRIOR ART

The industrial use of enzymes is currently generating much interestsince it is regarded as the prototype of a "soft" technology.

The types and number of industrial processes using enzymes are stilllimited. However, they are expected to grow. (See Ullmanns Enzyklopadieder technischen Chemie, vol. 10, pp. 522-526 ff., Verlag Chemie, 1975,and Kirk-Othmer, Encyclopedia of Chemical Technology, 3rd ed., vol. 9,pp. 173-224, John Wiley & Sons, 1980.)

Such processes are employed particularly in food and feedstufftechnology, in detergent technology and in the manufacture of leather,where the use of enzymes has long been a tradition. Enzymes are alsoused in selective chemical reactions; (See Raul Prawe, JahrbuchBiotechnologie, 1986-87, pp. 359-397, Carl Hanser Verlag, Munich.)Important examples of industrial enzymes (IE) aregamma-aminobutyrotransaminase, amylase, cellulase, collagenase, glucoseoxidase, glutamic acid decarboxylase, hemicellulase, invertase,catalase, lipase, pectinase, penicillase, protease, and streptokinase,among others.

The media in which the enzymes occurring in nature are active arepredominantly aqueous media, where a certain dependence on the pH valueand on the dissolved constituents of the aqueous medium is frequentlyobserved, and the aqueous medium may therefore be regarded as thestandard medium in enzymatic reactions.

Since enzymes are polypeptides whose activity depends primarily on theirstructural organization and which, like other proteins, can also bedenatured by certain organic solvents, caution has always been indicatedin the use of organic solvents together with enzymes.

In the recent past, the effect of additions of water-soluble organicsolvents to an aqueous medium has been determined, for example, in thecourse of investigations of immobilized enzymes, and it has beenobserved that as the dielectric constant decreases, the reaction rateincreases. (See H. Weetall and P. Vann in Enzyme Engineering, vol 4[ed., G. B. Brown et al.]; NCI Monograph No. 27 [ed., M. P. Stulberg],1967, pp. 141-152.)

THE OBJECT

In handling chemical agents, and especially in proportioning them, it isoften considered an advantage when they are present in liquid form. As arule, incorporating liquid preparations into liquid reaction mediaentails fewer problems than admixing powders or other solids. This istrue also of enzyme preparations.

This preference for working with liquid preparations can be observed inthe detergent industry, for example. On the other hand, liquid enzymepreparations is particular pose many problems. The most important ofthese has to do with the the stability of such enzyme preparations. Asmentioned above, enzymes in an aqueous medium are subject to theinfluence of other constituents of the medium, such as acids, bases,salts, surface-active and complexing components, other macromolecules,and particularly other enzymes, the substrates on which the enzymes act,and so forth

Such additives may have either a stabilizing or a destabilizing effect.As stabilizing additives for certain enzymes in aqueous solutions,glycols, polyglycols, surfactants, proteins and protein hydrolyzates,synthetic polymers, carboxylic acids, specific cations or anions and thelike have been proposed, for example. (See U.S. Pat. No. 4,519,934; L.Kravetz and K. F. Guin, Journal of the Am. Oil Chem Soc., vol. 62, 5,[1985 ]; L. Gianfreda, M. Modafferri and G. Greco, Enzyme Microb.Technol 7, 78-82, [1985]; K. Martinek and V. P. Torchilin, EnzymeMicrob. Technol. 1, 74-82 [1979]; U.S. Pat. Nos. 4,111,855 [1978],4,318,818 [1982], 3,557,002 [1971] and 4,169,817 [1979].)

The formation of inverse micelles in a water/surfactant/organic solventsystem has been taken into consideration as a further possible approachto the stabilization of enzymes in aqueous solution. (See P. L. Lisi,Angew Chemie 97, 449-460, [1985].)

The object of the present invention will now be explained in greaterdetail with reference to enzymes or enzyme systems used in themanufacture of leather.

Liquid proportioning has gained much ground of late over solidsproportioning especially in leather manufacture since it does indeedoffer advantages with regard to handling, for example. However, thepreparation of liquid preparations containing enzymes, such as enzymaticbating and soaking aids, poses the problem of insufficient stability ofthese products. This instability, particularly of pancreaticpreparations, is by no means an unexpected development but is alreadyknown to some extent from enzyme processing. In the case of proteases,allowance must also be made for the self-digesting action in particularand for the decomposing effect on other enzymatic proteins which arepresent. (See K. Martinek and V. P. Torchilin, Enzyme Microb Technol ,vol. 1, pp. 74-82 [1979].)

It should be noted that liquid enzyme preparations have failed so far togain commercial acceptance. Thus there has been a continuing need forliquid enzyme formulations (a) which are stable under the conditions ofuse, (b) in which the activity of the enzymes preferably is not affectedor not irreversibly affected, (c) which are compatible with the intendedend-use areas of the enzymes, and (d) which are economically andecologically acceptable. To be suitable for use with modernleathermaking methods, the loss of activity of such a so-called liquidenzyme should not exceed 15 percent over a period of from four to sixmonths.

THE INVENTION

It has now been found that the inventive liquid enzyme formulationssurprisingly meet practical requirements to a high degree. The inventionrelates to liquid enzyme formulations for technical use wherein thevehicle for the enzymes is at least one substantially anhydrous organicliquid or a mixture of such liquids. Suitable liquids for this purposeare liquids which are consonant with the above requirements, areadvantageously in common use, and preferably are ecologically safe andin particular substantially nontoxic and noninjurious to enzymes. Theorganic liquids to be used in accordance with the invention arepreferably hydrocarbons, but may contain oxygen or, less preferably,sulfur. Liquids of the formula (I)

    R.sub.1 --X--R.sub.2

are preferred, wherein, when X is carbonyl, R₁ and R₂ are the same ordifferent alkyl having 1 to 3 carbon atoms. If X is COO, then R₁ and R₂are the same or different alkyl having 1 to 6 carbon atoms or R₁ ishydrogen and R₂ is such alkyl. If X is oxygen or sulfur, R₂ is hydrogenor alkyl having 1 to 8 carbon atoms and R₁ may be alkyl having 1 to 8carbon atoms, or is ##STR1## in which R₃ is hydrogen, --CH₃ or --CH₂ OH,or R₁ is --(CH₂ --CH₂ O)_(n) --H, where n is an integer from 1 to 15.

R₁ and R₂ may also each be --CH₂ --CH₂ OH.

Further, R₁ and R₂, taken together with an oxygen or sulfur atomtherebetween, may form a 5- or 6- membered ring in which all ringmembers are --CH₂ --, except that one such ring member may be carbonyl(C═O) and one further such ring member may be oxygen.

Additional liquids include those of the formula (II) ##STR2## wherein R₅is --CH₃ or --CH═CH₂.

Illustrative of liquids of formula (I) are particularly carbonic esters,and more particularly cyclic carbonic esters such as ethylene carbonateand, specifically, propylene carbonate. Also of interest are polyhydricalcohols such as glycerol, ethylene glycol, butyl glycol, butyldiglycol, diethylene glycol, polyethylene glycols (MW at least 400),liquid polypropylene glycols, and etherified derivatives thereof;monovalent alcohols or fatty alcohols such as methanol, ethanol,isopropanol, n-butanol, isobutanol, 2-ethyl hexanol, and cyclohexanol;ethers such as diethylether, methoxypropanol, diethylene glycolmonomethyl ether, and particularly cyclic ethers such as dioxan andtetrahydrofuran; ketones such as acetone, ethylmethyl ketone, andcyclohexanone; esters such as ethyl acetate and butyl acetate; andlactones such as F-butyrolactone. Also to be mentioned are oxoalcoholsand their ethoxylation products having 3 to about 14 ethylene oxideunits; fatty alcohols and their ethoxylation products; olefins and theirsulfonated derivatives such as C₉ -C₁₂ -α-olefin sulfonate, for examplein the form of the sodium salt; and fatty alcohol ether sulfates such assulfated C₁₂ -C₁₅ -alcohols, condensed with ethylene oxide.

Further liquids are fatty acid alkanolamides, such as C₁₆ -C₁₈ -fattyacid ethanolamide; and alkylphenol compounds such as octylphenol andethoxylated derivatives thereof, such as nonylphenol ethoxylate with 8-9moles of ethylene oxide.

Further, mixtures of organic liquids can be of advantage.

Representatives of liquids of formula II are toluene and styrene. Also,turpentine, solvent naphtha, white spirit, aliphatic hydrocarbons, andpetroleum oils (boiling point range preferably between 50° C. and 180°C., and especially between 70° C. and 150° C.) are useful. With theexception of the polyethylene glycols, as a rule these are liquidshaving a molecular weight less than 110 and for the most part having aboiling point below 300° C. at normal pressure. (Further informationconcerning suitable liquids can be found in the monograph of Gnamm andFuchs, Losungsmittel und Weichmachungsmittel [Solvents andPlasticizers], 8th edition, Wissenschaftliche Gesellschaft, Stuttgart,1980).

The organic liquids to be used according to the invention areessentially anhydrous, i.e. as a rule they contain less than 1 percentby weight of water, and preferably not more than the usual mechanicallyheld moisture. Drying of the liquids is carried out conventionally, forexample by means of commonly used desiccants, dsitillation, includingazeotropic distillation, etc. (See Houben-Weyl, vol. I/2, pp. 765-886,Georg Thieme Verlag, 1959).

The presence of water in the preparations of the invention generally isnot contemplated, at least not in amounts that would be detrimental tothe purposes of invention. When liquids are used which do not mix withwater, the enzyme formulations may further contain surfactants.Preferred are nonionic surfactants of the type of the fattyalcohol/alkyl phenol ethoxylates (with oleocety; and olein-alcohol, forexample); moreover, adducts of polyglycols with from 4 to 42 moles ofethylene oxide, apart from anionic types such as alkyl sulfates, alkylether sulfates, alkyl phosphates, alkyl ether phosphates, alkylsulfonates and alkylaryl sulfonates. (See German Pat. No. 33 22 840.)The amount of the surfactants generally ranges from 0.001 to 50 weightpercent, and preferably from 0.005 to 20 weight percent, based on theliquid vehicle. The hydrophile-lipophile balance (HLB) of theemulsifiers (or the o/w emulsifying action) generally ranges from 8 to18, and preferably from 9 to 15. (With respect to emulsifiers and HLB,see Kirk-Othmer, 3rd ed., vol. 8, 910-916.) Also preferred are liquidswhich do mix with water, and especially liquids which are miscible withwater in any ratio. On first approximation, it should be assumed thatthe enzymes remain completely undissolved in the liquids. The tendencyto self-digestion or to decomposition of other enzymes thus is greatlyminimized.

The quantity of the liquids used is based mainly on practicality in thehandling of the enzymes and is not really critical. In general, theweight ratio of enzyme to liquid will not be less than 1:10; for reasonsof handling ease. For the sake of reasonable concentrations, the upperlimit of the weight ratio of enzyme to liquid will generally range from1:30 to 1:500. As a rule of thumb, a ratio of enzyme to liquid of from 1to about 50 has proved practical. Optionally, mixtures of the liquidsrecited above may be used. As the enzymes, which are essentiallyproteins, are added to the substantially anhydrous liquids, problemssuch as slow wetting and/or dispersal problems such as settling orcreaming may arise.

In developing the present invention further, it was found that theproblems arising in the preparation of homogeneous liquid enzymeformulations, such as creaming and settling, can be solved at least inpart by dispersing inorganic powdered additives in the liquids.

Powdered additions of inorganic dispersants have been described foraltogether different applications, namely, as dispersants foroil-in-water emulsions or as dispersants in free-radical beadpolymerization. (See Houben-Weyl, Methoden der organischen Chemie, vol.XIV/1, Macromolecular Substances, pp. 420-421, Georg Thieme Verlag,1961.) For example, insoluble salts of the alkaline-earth metals(carbonates, phosphates, sulfates and silicates), aluminum hydroxide,talc, bentonite, and, particularly, silicon dioxide, and especiallypyrogenic silicon dioxide (fumed silica). (See Kirk-Othmer, op. cit.,vol. 20, pp. 748-781; Ullmanns Encyclopadie der Technischen Chemie, 4thedition, vol. 18, pp. 652-656, Verlag Chemie 1979.)

The pyrogenic modifications of silicon dioxide are known to be preparedby gas-phase reactions, for example by flame hydrolysis or by use of anelectric arc. They generally consist of spherical particles having aprimary particle size from 5 to 500 nanometers. Their density is about2.2 g/cm³. Such materials are available commercially under the names"Aerosil" and "Cab-o-Sil".

Additionally, clays, particularly bentonite, are suitable for theaforementioned purpose, although attention must be paid that certainenzymes, for example pectinases, are adsorbed on bentonite with loss ofactivity.

Kaolin and bentonite are known to form thixotropic gels with a number ofpolar and nonpolar organic liquids. The thixotropy of the gelsdisappears upon the addition of minor amounts of longer-chain alcoholsfor example. (See Ullmanns Enzyklopadie der technischen Chemie, 4th ed.,vol. 23, pp. 318-326, Verlag Chemie, and U. Hoffmann et al., Kolloid-Z.151, 97-115 [1957].)

By clays are meant the usual aluminosilicates, and by bentonites thecommercial preparations consisting mainly of montmorillonite (Al₂O₃.4SiO₂.H₂ O). Of special interest are organically activatedbentonides, for example, those formed by reaction of sodiummontmorillonite with quaternary alkylammonium compounds. Through cationexchange, so-called organophilic bentonites, which swell in organicliquids, are formed. (See Ullmanns, loc. cit., p. 323.) As a rule, thecommercial grades (flakes, particle sizes ranging from 0.5 to 5 microns)may be used. However, it is advisable to subject them to a treatmentwith shearing action in the liquid selected. As a rule, the liquidsshould contain from 0.1 to 3 weight percent, and preferably from 0.3 to1 weight percent, based on the liquid, of inorganic additives. It hasproved particularly advantageous to subject the inorganic additi.on tothe enzyme concentrate, together with the liquid, to shearing action inappropriate equipment, and particularly in a dispersion mill. The lattermay be of the commercial type. (See Ullmanns Enzyklopadie dertechnischen Chemie, 4th ed., vol. 1, p. 239, Verlag Chemie, 1972.)

The rotational speed and the treating time depend mainly on the type ofthe equipment. A treating time of from 2 to 60 minutes at a peripheralspeed of from 4 to 36 meters per second will serve as a guide for theaction of commercial dispersing apparatus on bentonite in one of theliquids to be used in accordance with the invention.

Propylene carbonate has been found to be a particularly effectiveliquid, especially in combination with silicon dioxide or withbentonite.

So far as is known, the formulations of the invention are not subject toand, restrictions with respect to the enzyme component, except insofaras the stability of the enzymes themselves to organic liquids such asalcohols is concerned. A requirement is that the organic liquid vehiclemust be compatible with the end use of the enzymes. It was not to beforeseen that, by the use of inorganic dispersing agents, organic liquidcarrier systems for enzymes could be prepared, which systems would ingeneral more closely approach the ideal requirements of technology, notonly from the viewpoint of physical and biological stability, but alsofrom the point of view of ready availability.

To enchance the anti-settling or anti-creaming effect, weakly polarorganic liquids may be added to the vehicles, especially when the latterare polar liquids of the type of formula (I). Thus, it may beadvantageous if the vehicle contains from 1 to 10 percent by weight ofhydrocarbons, and particularly branched or linear aliphatics having from5 to 20 carbon atoms. Illustrative of these are petroleum fractions inthe boiling range of approximately 50° to 180° C. See also the abovedefinition of liquid.)

The enzymes are primarily those which are already being usedindustrially or in other fields of application. (See "The Prior Art"above.)

(A) Proteases (EC 3.4; Kirk-Othmer, loc. cit., vol. 9; Aunstrup in"Industrial Aspects of Biochemistry" (B. Spencer, ed.), vol. 30 (I), pp.23-46, North Holland, 1974).

(a) Animal-derived, for example:

(a₁) Rennin (EC 3.4.23.4.)

(a₂) Pancreatic proteases:

Pancreatin, and particularly trypsin and chymotrypsin (optimum pH rangeabout 7 to 10);

Pepsin (EC 3.4.23.1) (optimum pH range about 1.5 to 4.0;

Cathepsin (EC 3.4.23.5) (optimum pH range about 4.0 to 5.0).

(b) Plant-derived:

(b₁) Papain (EC 3.4.22.1) (optimum pH range about 5.0 to 8.0);

(b₂) Ficin (EC 3.4.22.3) (optimum pH range about 4.0 to 9.0);

(b₃) Bromelain (EC 3.4.22.4 and 3.4.22.5) (optimum pH range about 5.0 to7.0.

(c) Microbially derived (see L. Keay in "Process Biochemistry", 1971,17-21):

(c₁) From bacillus species, for examples, B. subtilis, B. licheniformis,B. alkalophilus, B. cereus, B. natto, B. vulgatus, B. mycoides.

(c₂) From streptococci.

(c₃) From streptomyces, for example, Streptomyces fradiae, S. griseus, Srectus.

(c₄) From aspergillus species, for example, Aspergillus flavus-oryzae,A. niger, A. saitoi, A. usamii.

(c₅) From members of the genera Mucor and Rhizopus, for example, Mucorpusillus, M. mietrei.

(c₆) From endothia strains, for example, E. parasitica.

(c₇) From trametes strains, for example, Trametes sanguinea.

Enzymes are classified not only according to their source but also onthe basis of the site of attack (exoenzymes vs. endoenzymes) and of theactive site of the proteases (serine proteases, which are inhibited bydiisopropyl fluorophosphate [DFP]; sulfhydril enzymes).

A factor that is of considerable practical importance is that enzymeactivity is a function of pH.

From a practical point of view, proteases are therefore classed asfollows:

(i) Alkaline proteases with optimum activity in the pH range of about7.5 to 13, and particularly alkaline bacterial proteases (EC 3.4.21),most of which are of the serine type, and alkaline fungal proteases.

(ii) Neutral proteases with optimum activity in the pH range from 6.0 to9.0, and particularly neutral bacterial proteases (EC 3.4.24), whichbelong to the metalloenzymes, and fungal proteases such as bacillusproteases, pseudomonas proteases, streptomyces proteases, andaspergillus proteases.

(iii) Acid proteases with optimum activity in the pH range from 2.0 to5.0 (EC 3.4.23), and particularly acidic fungal proteases, for example,from Rhizopus species, Aspergillus species, Penicillium species, Mucorspecies, as well as Impex lacteus and Endothitia parasitica.

Proteases are used industrially in leather manufacture, in detergentsand in cleansing, in desizing, in cheesemaking, in the tenderization ofmeats and in the stabilization of beer, for example.

Examples of alkaline proteases are, in particular, the subtilisins,alkaline bacterial proteinases of the serine type, which in the pH rangefrom 9 to 10 are stable and relatively insensitive to perborate.

The proteolytic activity of enzymes is usually determined by the Ansonhemoglobin method (M. L. Anson, J. Gen. Physiol. 22, 79 [1939]) or bythe Lohlein-Volhard method ("Die Lohlein-Volhard'sche Methode zurBestimmung der proteolytischen Aktivitat" in GerbereichemischesTaschenbuch, Dresden/Leipzig, 1955) and in that case expressed in LVU(Lohlein-Volhard units).

An LVU is the amount of enzyme which under the specific conditions ofthe method digests 1.725 mg of casein. For determination of the activityof enzymes active in the acid range, units are also used in what followswhich are derived from the Anson method. These nits are known as"proteinase units (hemoglobin)", or U_(Hb). One U_(Hb) corresponds tothe amount of enzyme which catalyzes the release of fragments soluble intrichloroacetic acid from hemoglobin equivalent to one micromole oftyrosine per minute at 37° C. (measured at 280 nm). (1 mU_(Hb) =10⁻³U_(Hb).)

(B) Amylases (EC 3.2; see Ullmanns, loc. cit., vol. 10, pp. 506-510, and"Industrial Aspects of Biochemistry" (B. Spencer, ed.), loc. cit., pp.143-144, 175).

(a) Endoamylases:

(a₁) alpha-Amylases (alpha-1,4-glucosan hydrolases) (EC 3.2.1.1)

(a₂) alpha-1,6-glucosan hydrolases

(b) Exoamylases: (saccharogenic amylases):

(b₁) beta-Amylases (alpha-1,4-glucosanmaltohydrolases) (EC 3.2.1.2)

(b₂) Glucoamylases (alpha-1,4-glucosanglucohydrolases) (EC 3.2.1.3)

Alpha-amylases are known to occur in plants, for example, together withbeta-amylases. They are commercially produced from pancreas and frombacterial and fungal cultures.

They are obtained most readily from bacillus pecies such as B. subtilis,B. mesentericus, B. polymixa, B. amyloliquefaciens and B. licheniformis;from fungi, and particularly from aspergillus species such as A. niger,A. phoenicis, A. oryzae and A. awamori; from Mucor strains such as M.rouxianus; from Rhizopus strains such as R. delemar, R. oryzae and R.japonicus; and from endomyces strains such as E. fibuliger. The pHoptimum of alphaamylases is predominantly in the range from 4.7 to 7.2.Amylases find use in the food industry (see "Biotechnology" [H.-J. Rehm& G. Reed, ed.], Vol. 5, Verlag Chemie, 1983; "Industrial Aspects ofBiochemistry" [B. Spencer, ed.], vol. 30, part I, pp. 139-186 and213-260, Elsevier [1973]), in the liquefaction of starch, in maltproduction, in the production of ethanol, in desizing, in leathermanufacture, etc.

The activity of alpha-amylases can be determined, when starch is used asthe substrate, by the method of Sandstedt, Kneen & Blish (Cereal Chem.16, 172 [1939] and Technical Bulletin No. 1024, U.S. Department ofAgriculture). One amylase unit (=one SKB unit) is the amount of enzymewhich at 30° C. and under the other conditions specified is able todextrinate one gram of soluble starch in one hour.

The Willstatter method is used to determine the activity of pancreaticamylase. (Hoppe-Seylers, Z. physiol. Chem. 126, 143 [1923].) AWillstatter amylase unit is defined as 100 times the amount,of enzymewhich under the test conditions specified breaks down starch at such arate that the monomolecular reaction constant is 0.01.

(C) Lipases (EC 3.1.1.3)

As is known, lipases are carboxyl esterases which cleave glycerol esterin aqueous emulsion. Thus they differ from other carboxyl esteases whichattack the substrate in aqueous solution.

(a) Pancreatic lipases

The pancreatic enzyme complex contains, apart from lipases, mostlyesterases as well as proteases and amylases as commercially importantcompanion enzymes. The pH optimum (for olive oil) is in the range from 7to 8.5, with the range of activity extending from pH 6.5 to 9.5.

Lipases are generally highly unstable, especially to proteolyticbreakdown by companion proteases.

(b) Microbiological lipases, for example, from Pseudomonas fragii,Aspergillus species (e.g., A. luchuensis, Candida cylindracea,Geotrichum candidum, Humicola lanuginosa, Mucor pusillus, Penicilliumspecies (e.g., P. chrysogenum, P. oxalicum), and Rhizopus species (R.nigricans, R. oryzea).

These lipases generally have at least one pH optimum at a pH above 7.0.To the extent that their instability permits it, lipases find use inwaste disposal, in leather manufacture and in the food industry, forexample.

(D) Catalases/hydroperoxidases (EC 1.11.1.6):

(a) From animal tissue, for example, from liver.

(b) From plants, for example, from horseradish.

(c) From microorganisms, for example, from Micrococcus lysodeicticus.

Catalases are used in peroxide bleaching and in the milk industry.

(E) Cellulases (EC 3.2.1.4) [See Ullmanns, loc. cit., pp. 510-511.)

As is known, cellulase is an enzyme complex whose componentssuccessively take part in the breakdown of native cellulose.

Cellulases are found in insects, mollusks and microorganisms (bacteria,mold fungi). Commercially utilized sources of cellulases areparticularly aspergillus, neurospora, rhizopus, trichoderma and trametesstrains.

Commercial preparations have optimum activity between pH 4 and 6.Prior-art cellulase preparations lose approximately 10 to 20 percent oftheir activity per year.

Cellulases find commercial use in the food industry for conversion ofcellulose-containing wastes, etc.

The presen invention will now be described in greater detail withreference to the use of enzymes in the manufacture of leather. The useof enzymes, and particularly of proteolytic enzymes, has been part andparcel of leather-making, and particularly of the beamhouse processes,since the introduction of the enzymatic bate and tryptic digestiveenzymes from the pancreas in the "Oropon®" bate by Dr. Otto Rohm (GermanPat. No. 20 05 19) some 80 years ago.

In addition to being used in bating (German Pat. Nos. 9 27 464, 9 76107, 9 41 811, 9 74 813, 9 75 095, 9 76 928, 11 20 066, 11 34 474, 12 19620 and 12 82 837 and U.S. Pat. Nos. 3,939,040 and 4,273,876, enzymepreparations are used also in soaking (German Pat. Nos. 2 88 095, 9 76602, 10 22 748, 10 34 317, 12 82 838 and 20 59 453, and U.S. Pat. Nos.4,278,432 and 4,344,762); in hair loosening and opening up (U.S. Pat.No. 4,294,087); in unhairing (German Pat. Nos. 10 26 038, 12 11 349, 1155 560, 12 30 169 and 12 88 728, and U.S. Pat. No. 3,623,950); in liming(German Pat. Nos. 10 23 183, 12 03 416, 20 53 016 and published Germanpatent application No. OS 34 29 047); in pickling (German Pat. Nos. 8 47947 and 9 41 680) or in a compact process (U.S. Pat. Nos. 3,986,926 and3,966,551); in wet degreasing (German Pat. No. 33 12 840); for looseningthe fibrous structure of furs (U.S. Pat. Nos. 3,549,495 and 3,558,430),etc. Enzyme preparations are further used to dissolve untanned machinetrimmings and other byproducts of leather manufacture (U.S. Pat. No.4,210,721, Swiss Pat. No. 631,486, U.S. Pat. Nos. 4,293,647 and4,220,723), keratin-containing raw stock (U.S. Pat. No. 4,232,123),elastincontaining preparations (U.S. Pat. No. 4,179,333); for working upcollagen-containing raw stock (U.S. Pat. No. 4,220,714), etc.

Consistent with the aforesaid enzymatic processes, liquid enzymeformulations in accordance with the present claims may be used in thevarious operations of leather manufacture (see Ullmanns Enzyklopadie dertechnischen Chemie, 3rd ed., vol. 11, p. 609, Urban/Schwarzenberg;Ullmanns Enzyklopadie der technischen Chemie, 4th ed., vol. 16, pp.111-174, Verlag Chemie, 1978; F. Stather, Gerbereichemie undGerbereitechnologie, 4th ed., Akademie-Verlag, Berlin, 1967), namely:

(I) in soaking,

(II) in hair loosening, liming and unhairing,

(III) in deliming and bating, and optionally

(IV) in pickling.

Usually the liquors are in the range of from 50 to 500 weight percentbased on the weight of the hides and skins used in the respective steps.

(I) Soaking

The soaking of hide stock, in which the hardening of the hides, orskins, resulting from salt curing is reversed, is usually carried out ata pH between 7.0 and 10.0. The concurrent use of enzymes, andparticularly of proteolytic enzymes (see [A] above), accelerates thesoftening action through "digestion" of the water-soluble and otheralbumins which are not a part of the collagenous fiber structure of thehide.

In general, enzymes whose range of activity (or pH and 10.0 are employedin soaking. Revival of the noncollagenous albumins assures faster andmore intensive wetting of the hide.

The soak water is advantageously made slightly alkaline; however, the pHvalue should always remain below 12. The use of soaking aids (such asmonoethanolamine with an antiseptic such as zephyrol ornaphthalenesulfonic acid in combination with substituted phenols; highlysulfonated ricinoleic acid butyl ester with a methylcyclohexanolmixture; fatty alkyl sulfates with solvents) is also advantageous.Examples of suitable enzymatic additives for the inventive liquid enzymeformulations are the proteases listed under (A) above, and particularlythose named under (A) (c), and more particularly microbial proteasesactive in the pH range from 4 to 9.5, especially fungal proteinases fromaspergillus species such as A. saitoi and A. usamii, for example, andparticularly acid proteinases with activity in the pH range from 2.5 to4.5; also those from A. oryzae active in the pH range from 4.0 to 9.5,and those from A. niger and A. flavus active in the pH range from 9.5 to11.0.

The concentration of proteolytic activities of the proteinases usedgenerally ranges from 0.1 to 1.0 Anson unity, or from 1,000 to 3,000LVU, per liter of soak liquor.

The soak liquors may further contain amylases according to (B) above.Amylases occur as companion enzymes of fungal proteinases. They promotethe cleavage of glucosidic bonds in the proteoglycans and glycoproteinsof the hide.

Well suited are amylases of microbial origin, and more particularlythose from aspergillus species such as A. oryzae and A. niger,especially those active in the pH range from 3.0 to 5.8. Suited for useare also those of bacterial origin, for example, those derived fromBacillus subtilis, B. mesentericus and B. polymixa with activity in thepH range from 5.0 to 7.0.

As a rule, the glycolytic activity of amylases ranges from 500 to 2,000SKB.

The temperature of the soak liquors is advantageously higher than 20° C.The soaking duration should be as short as possible and generally isbetween 4 and 36 hours.

(II) Hair loosening, liming, opening up

For unhairing, lime liquors are used most often. (See Ullmanns, loc.cit., 3rd ed., vol. 11, p. 560; 4th ed., vol 16, pp. 118-119.) So-calledsharpened lime liquors, preferably a combination of calcium hydroxideand sodium sulfide, are used throughout and in the presence ofbuffering, swelling-inhibiting liming.aids such as wetting agents, andparticularly cationic wetting agents, in combination withmonoethanolamine and disinfectants, for example, quaternaryalkyldimethylbenzylammonium compounds, or dialkylamine and its sulfate.For hair loosening and opening up, enzymes which in this pH range remainsufficiently stable may be used is addition to the usual limingchemicals. Soaking and liming may be combined by gradually increasingthe pH value and using appropriate enzymes.

The use of enzymes in conjunction with the liming/hairloosening/unhairing operations generally takes place in the pH rangefrom 9 to 11, and more particularly from 9 to 12.

Consistent with German Pat. No. 29 17 376 or U.S. Pat. No. 4,294,687,respectively, the hide, freed of curing salt, may first be pretreated inthe acid pH range with substances cleaving disulfide bridges, and thenhair loosening and opening up may be brought about simultaneouslywithout prior soaking by the use of proteases active in the alkaline,range at a pH of about 11 to 13. This is then followed by the furtherprocessing steps (III) and optionally (IV) usually carried out in thebeamhouse. It is advisable to use in the beamhouse operations (II)alkaline bacterial proteinases (serine proteases), for example, from B.subtilis, B. licheniformis, B. firmus, B. alcalophilus, B. polymixa andB. mesentericus.

These proteases generally have an activity ranging from 8,000 to 10,000LVU per gram. They are advantageously used in amounts of from 0.1 to 10weight percent, and preferably from 1 to 5 weight percent, based on theweight of the salted hides and skins (raw weight).

The enzymatic reaction in unhairing and opening up is carried out atabout 18° to 28° C. The reaction time generally ranges from 12 to 24hours, and more particularly from 12 to 16 hours.

Unhairing or dewooling can also be performed with alkaline fungalproteinases of the above type (i), for example, with aspergillusproteases, and particularly with those from A. niger and A. flavus whichare active in the pH 9.5 to 11.0 range.

Moreover, the enzymatic unhairing method of German Pat. No. 34 29 047,in which hides and skins are treated in a liquor in the pH range from 9to 11 with proteolytic enzymes having optimum activity in the pH rangefrom 2 to 7.5, may be employed, unhairing being then carried out. Theenzymes used then are proteases of the above type (iii), andparticularly from A. oryzea, A. saitoi, A. parasiticus, A. usamii and A.awamori, from Paelomyces species, Penicillium species or also Rhizopusspecies and/or from Mucor pusillus, as well as the acid proteases listedunder (A) (a) and (A) (b) above.

As a rule, from 0.5 to 6 weight percent, and preferably from 1 to 3weight percent, based on the weight of the salted hides or skins, isused. The activity generally ranges from 50 to 200 mU_(Hb).

(III)

Deliming and bating are preferably carried out with the aid of enzymes.Deliming serves to reduce the alkalinity of the pelts from a pH valuebetween 13 and 14 to a pH of from 7 to 8. Deliming is preferably done,not with strongly dissociated but with weak organic acids of the type ofthe dicarboxylic acids, or then with weakly acid salts. In bating,residues of epidermis, hair and pigments should be removed and furtheropening up should be brought about. Moreover, noncollagenous albuminconstituents should be removed. (See Ullmanns, 4th ed., vol. 16, loc.cit., pp. 119-120.) Bating is carried out conventionally at pH 7.5 to8.5. The use of cyclic carbonates in deliming is known from German Pat.No. 31 08 428.

The duration of the bate as a rule is between 1 and 6 hours, preferably1-2 hours as a short bate. As a rule of thumb, the proteases can be usedin such amount that from 1 to 10 Lohlein-Volhard units are prevent pergram of pelt weight.

The temperature is advantageously between 25° C. and 35° C., preferably30° C. Any conventional commercial tanning vessel, such as a drum,paddle-vat, mixer, or tanning machine can be used. (See O'Flaherty etal., The Chemistry and Technology of Leather, vol. 1, ACS MonographSeries, Reinhold Publishing Corporation, New York 1956).

Lipases according to (C) above, for example, pancreatic lipasesexhibiting activity in the pH 7.0 to 9.0 range, may be used concurrentlyin bating.

Amylases according to (B) above, for example, pancreatic amylases whichare active at a pH of from 5.5 to 8.5 and promote the cleavage ofglycoside linkages in bating, will also exert a beneficial influence onbating, particularly as companion en of trypsis and chymotrypsin.

(IV) Pickling

To prepare the pelt for mineral tanning, it has to be acidified, thatis, its pH has to be reduced from about 8 to the 3 to 4 range. This isdone in the pickle liquor, which is an acid/salt solution in water, forexample, sulfuric acid or formic acid together with sodium chloride.

Recommended are mold tryptases, pancreatic tryptases and bacterialtryptases, for example, optionally together with carbohydrate-splittingenzymes, derived in particular from bacteria or from mold fungi.

ADVANTAGES

The inventive liquid enzyme formulations offer a generally applicablesolution, not likely to give rise to difficulties, to the problem ofmaking use of liquid enzyme formulations, which is often desirable.Being able to combine enzymes with other components which in an aqueousmedium and on prolonged exposure would interfere with enzyme activity isa further advantage. This is true particularly of the ability to combinedifferent enzymes, for example, proteases, not only with amylases,lipases, etc., but also with hydrotropes such as urea, guanidine salts,cumene sulfonate, etc.

More in particular, the following advantages of the liquid preparationsof the invention over powdered preparations are evident:

Economic advantages: water soluble salts such as ammonium sulfate orsodium sulfate which are commonly added to powdered enzymes as diluentsand/or stabilizes can be dispensed with in the preparations of theinvention. Non-liquid components amount to only a fraction of thosepresent in conventional preparations. The salt content of prior artproducts, further, is responsible for a number of leather imperfectionswhich, thus, can be avoided using the new preparations.

Ecological advantages: the loading of waste water with salts and thelike, which represents a considerable source of pollution, for the mostpart disappears. As surface active agents, biologically degradeableagents are preferably employed.

Greater applicability: The invention permits the combination of enzymeswith one another, with further treating agents, with activators, and thelike, which are incompatible in aqueous concentrates. In contrast toaqueous enzyme preparations, the enzyne conccntration in thepreparations of the invention can extend over a wide range.

Extreme stability: to the limit of current experience, the preparationsof the invention are very stable. For instance, a combination of fungusproteases and pancreatic enzymes is still stable after 6 months'storage. The danger of contamination, for instance with microorganisms,is minimized. The need for anti-fungal or anti-bacterial protection isunnecessary. In aqueous liquid enzyme preparations, the stabilizingagents which are employed adversely affect enzyme activity.

Uniform enzyme activity: experience, especially in the area ofconventional leather preparation, has shown that the use of enzymesleads to defects in the product if the enzyme activity is non-uniform,as, for example, in the use of solid enzyme preparations. The liquidpreparations of the invention can be dispersed more uniformly and morerapidly.

Safety considerations: in contrast to powdered preparations, the liquidformulations of the inventions are far superior from the point of viewof safety, since there is no development of dust and no provocation ofallergies. In comparison with aqueous preparations, there is theadvantage that in case of spills, leaks, splashes, and other suchaccidental loss of the enzyme preparation, the enzyme is not immediatelyactive but, as a rule, becomes so only upon the addition of water.

Dosing advantages: because of the stability of the enzyme in an organicmedium, constant enzyme activity can be assumed. Hence, measurement ofthe preparation with the necessary accuracy can be by volume,eliminating the need for inconvenient weighings in order to establishthe required enzyme concentration.

Compatibility with auxiliaries: auxiliaries desired in the finalproduct, such as builders, polyphosphates, or zeolites, can be dispersedin the liquid organic carrier together with the enzyme, which is notpossible using an aqueous phase.

Stability to creaming and settling: in contrast to enzyme preparationsin which, for example, a crystalline enzyme is preserved in an organicliquid such as toluene, whereby the enzyme, for example a pancreaticenzyme, usually settles on the floor of the container, the inventionmakes available stable enzyme dispersions in which the stabilizer in noway adversely affects the enzyme activity. In contrast, the stabilizerin aqueous liquid formulations as a rule considerably degrades enzymeactivity.

The examples which follow will serve to elucidate the liquid enzymeformulations of the invention and their use.

An asterisk at the solvent shall indicate, that it is miscible withwater at all proportions at room temperature.

EXAMPLES Example A

Preparation of a liquid enzyme preparation based on pancreatic andfungal enzymes

700 g of propylene carbonate (0.6% water) to which 3.5 g of anorganically modified bentonite (e.g., "Bentone 27", marketed by KronosTitan, Leverkusen) has been added are dispersed for 45 to 60 minutes ata rotative speed of 16 meters/second in a toothed-disk agitator. (Disk:tank=1:2.5; the filling height is from 2 to 21/2 times the diskdiameter; the spacing of the agitator from the floor is one-half thedisk diameter.)

With further dispersing, 2.54 g of a fungal protease concentrate derivedfrom Aspergillus parasiticus (150,000 LVU; pH optimum, 7.5 to 9) and1.48 g of a pancreatic enzyme preparation (220,000 LVU; pH optimum, 6 to8) are added. Dispersing is then continued at constant rotative speed,care being taken to not exceed a temperature of 40° C. The end producthas an activity of 1,000 LVU and exhibits no loss of activity after 4weeks at 20° C. No creaming or settling of the enzyme is observed in theproduct.

The dispersing conditions (rotative speed) and the type of theorganically modified bentonite have to be adjusted according to the typeof the solvent, the particle size, and the concentration of the enzymeconcentrate.

Example B

Combined deliming and bating with a liquid enzyme product based onpancreatic and fungal enzymes in propylene carbonate.

10 kg limed and washed bovine pelts; split thickness, 3 to 4 mm; 30%water; temperature, 35° C.; 3% enzyme solution comprising pancreatic andfungal enzymes in propylene carbonate as in Example A (activity, 1,000LVU/g), 0.2% nonionic wetting agent based on fatty alcohol ethoxylatewith from 8 to 9 moles of ethylene oxide.

After 80 minutes, the pelts are completely delimed throughout, and thescud is thoroughly loosened. The final pH of the liquor is 8.0. No toxichydrogen sulfide escapes during deliming since the pH value never dropsbelow 9.00.

Example C

920 g of butyl glycol and 40 g of petroleum, boiling point 110 to 130,to which 30 g of an organically modified bentonite (e.g., "Bentone 27"of Kronos Titan, Leverkusen) has been added are dispersed for 38 minutesin an Ultra-Turrax agitator at 16 meters/second.

The dispersion is allowed to stand for 24 hours. Under the abovedispersing conditions, 18.5 g of a neutral protease from a Bacillussubtilis strain (70,000 LVU/g; pH optimum, 5.5 to 7) is added.Dispersing is continued for another 5 minutes. The end product has anactivity of 1,300 LVU. After 5 weeks, no loss of activity is evidentfrom the LVU measurement. The liquid enzyme formulation remainshomogeneous; there is no settling or creaming.

Example D

Enzymatic soaking and decreasing of cattle hides soaked for dirt removal

Percentages based of salted weight:

100.0 kg cattle hides soaked for dirt removal

200.0% water at 26° C.

0.8% liquid enzyme formulation from Example C

0.4% nonionic wetting agent based on fatty alcohol ethoxylate with 6moles of ethylene oxide

0.2% of a chloroacetamide-based bactericide

After a soaking time of 5 hours, the hides have been completely soakedback and are at the same time ready for hair loosening. A large portionof the natural fat in the hides is emulsified in the soak liquor.

The hides soaked in the above manner are limed by prior-art methods andprocessed further into crust leather for shoes.

Example E

1,000 g of petroleum, boiling point 110° to 130° C., is heated to 40° C.and after the addition of an organically modified bentonite (e.g.,"MPA-X-2000" of Kronos Titan, Leverkusen) dispersed for 10 minutes. Then2.5 g of an acid fungal protease from Aspergillus parasiticus (80,000LVU/g; pH optimum, 3.5 to 5) are added and dispersing is continued foranother 5 minutes.

A liquid enzyme formulation having an activity of 200 LVU is obtainedwhich after 4 weeks at 20° C. exhibits no loss of activity. The solutionis homogeneous and neither creams nor settles.

Example F

Degreasing and enzymatic loosening of pickled sheep and lamb pelts

Drumming

(Percentages based on pickled weight)

200% of water at 35° C., 15% of sodium chloride, keep in motion for 5minutes, add 10 kg of pickled pelts, and keep in motion for 30 minutes;flesh; drain liquor.

Degreasing

(Percentages based on fleshed weight)

30% of water at 35° C., 2% of a nonvolatile wetting agent based on fattyalcohol ethoxylate (6 to 8 moles of ethylene oxide), 6% of the liquidenzyme from Example E (200 LVU/g), 8 kg pickled pelts, keep in motionfor 30 minutes.

The pelts are well degreased and loosened due to the action of theenzyme/surfactant/petroleum combination.

For depickling, commercial depickling tanning agents may be added to areplenished or fresh liquor and the stock may then be processed furtherconventionally.

Example G

927.5 g of a non-ionic anhydrous wetting agent, prepared from nonylphenol plus 8.5 mol of ethylene oxide and 12 g of fumed silica (Aerosil380® product of of Degussa, Hanau, West Germany) are mixed together with5 g of a surface active agent (Borchigen STL®, based on modified fattyacid, product of Borchers GmbH, Goslar, W.-Germany) in a stirringvessel. The mixture is dispersed for 15 minutes with a toothed-diskagitator at a rotative speed of 15-18 m/sec. The original viscosity of15 mPas reaches 255 mPas, viscosity is determined using a Brookfieldviscometer condition I/6 upm.

With further dispersing 55 g of α-amylase 90 000 SKB/g (for SKB-unitsand analysis see R. M. E. Sandstedt, E. Kneen and M. J. Blish, CerealChem. 16, 712 (1939) Technical Bulletin No. 1024 U.S. Department ofAgriculture) are added. After 15 minutes of further dispersing oneobtains a liquid enzyme formulation with an initial activity of 5 000SKB/g. No creaming or settling of the enzyme is observed after 4 weeks'storing in the product. The loss of activity after storage at 25° C. is3.4%.

Example H

Enzyme supported prewashing of jeans fabrics.

10 kg of denim cloth (jeans fabric) which had been sized with starch areput into 50 l of water at 40° C. to which 50 ml of the liquid enzymeformulation of example G are added in a washing machine. The temperatureis then raised to 60° C. and the cloth is washed for 5-10 minutes and istwice rinsed afterwards with 50 l of water each time at 40° C.

An additional washing step using a wetting agent to dispose of degradedstarch turned out to be not necessary. The cloth that has been desizedin this way has a pleasant, soft feel.

Example I

Liquid, biodegradable heavy-duty detergent

466 g of 1,2-propylene glycol and 400 g of nonionic surfactant(Marlipa®013-90, isotridecyl alcohol plus 9 moles of ethylene oxide,product of Chemische Werke Huls AG, W.-Germany), 100 g of ethyl alcohol(98%) plus 10 g of fumed silica (Aerosil 380®, product of Degussa AG,W.-Germany) and 4 g of a surface active agent (modified and etherifiedfatty acid Borchigen STL®, product of Borchers GmbH, Goslar, W.-Germany)are mixed and dispersed in a stirring vessel using a toothed diskagitator for 15 minutes at a rotative speed of 15-18 m/sec. Theviscosity of the liquid was found to be 1 250 mPas. The followingcomponents are added and are actively dispersed in the order

6 g of alkaline protease from Bacillus subtilis (activity 92 000 LVE/g)

4 g of amylase from B.subtilis (activity 90 000 SKB/g)

6 g of an optical brightener (4,4'-bis-(4-anilino-6-[N-(2-hydroxyethyl)-N-(2-carbamoylethyl)-amino]-s-triazine-2-yl-amino-2,2'-stilbene-disulfonicacid

2 g of ethylene diamino tetraacetic acid, sodium salt (sodium salt ofEDTA)

6 g of -C₉ -C₁₂ olefinsulfonic acid, sodium salt (98%)

After addition of the components the mixture is exposed to dispersionconditions for another 15 minutes. A viscous yet readily pourablesolution is obtained, which when stored for 4 weeks at 25° C. showsneither creaming nor settling. The α-amylase showed a loss of <1% of theoriginal activity. The loss of activity of the protease was found to be1.4%.

Example J

Washing of a blood stained cotton fabric at 60° C.

500 g of a white cotton fabric which had been soaked with bovine bloodand dried was treated with 6 g of the enzymatic detergent of Example I(which had been stored for 3 month before use), in 2.5 l of water and 2g of sodium metasilicate for 15 minutes at 60° C. Then the fabric waswashed two times with 2.5 l of water at 30° C. For comparison theidentical fabric stained in the same way was treated with 6 g of aliquid detergent which was identical to that of Example I, except that1,2-propylene glycol had been replaced by water.

Evaluation of the result of washing in both cases on the remissionphotometer ELREFOMAT DFC 5® (Zeiss, W.-Germany) showed that treatmentwith the anhydrous detergent preparation yielded a desorption of bloodfrom the fabric that was 46% higher than with the aqueous preparation.

Example K

Preparation of a liquid lipase preparation

In a vessel, 968 g of a C₁₆ -C₁₈ fatty alcohol reacted with 5 mol ofethylene oxide (Genopal 0-050®, product of Hoechst AG, West-Germany), 12g of fumed silica (Aerosil 380®, product of Degussa AG, W.-Germany) and4 g of a surface active agent (Borchigen STL®) are dispersed by means ofa toothed-disk agitator at a rotative speed of 15-18 m/s for 15 minutes.The viscosity of the system rises from 18 mPas to 290 mPas. Then 16 g ofa lipase enzyme concentrate from Pseudomonas sp. with an activity of 5000 LCA/mg (for definition and determination of LCA-units c.f. Semeriva,Biochem. 10, 2143 (1971) are added and the mixture is dispersed foranother 10 minutes. One obtains a liquid enzyme formulation with anactivity of 80 LCA/mg, which after 6 weeks at 25° C. does not show anycreaming or settling. The activity of the formulation was found to be77-78 LCA/mg.

Example L

Saponification of beef suet

400 g of beef suet and 600 g of water are emulsified with 5 g of thelipase preparation of Example K at 50° C. and the emulsion is stirred atthis temperature for 3 hours. Then the proportion of fatty acidsliberated in the process of saponification is determined by titrationaccording to IUPAC-standard, Methods of Oils, Fats and Derivates, 6thEd., pg. 56, Pergamon Press, Oxford (1979). The degree of hydrolysis wasfound to be 72%.

Example M

Preparation of a liquid cellulase-hemicellulase formulation

882 g of ethylene glycol, 13 g of fumed silica (Aerosil 200®, product ofHoechst AG, W.-Germany) and 5 g of a surface active agent based onmodified fatty acid (Borchigen STL®, product of Borchers GmbH, Goslar)are dispersed in a stirring vessel using a toothed-disk agitator at arotative speed of 15-18 m/sec for 10 minutes. The viscosity of thesystem rises from 35 mPas to 330 mPas. Then 100 g of an enzymeconcentrate of cellulase/hemicellulase from Trichoderma viride with anactivity of 10 000 FPU/g (FPU units as defined by M. Mandels, R.Andreotti and C. Roche, Biotech. Bioeng. Sym. 6, 17 (1976) are added andare dispersed for another 10 minutes.

One obtains a viscous, homogeneous enzyme preparation (viscosity 2 600mPas) with an activity of 1 000 FPU/g), which after 5 weeks storage at25° C. shows no sign of settling or creaming.

Example N

Use of cellulase-hemicellulase formulation in gluten filtration

10 kg of a glutene suspension (dry matter contents about 30%) which wasseparated from corn starch by centrifugation is treated with 3 g of thecellulase preparation according to example M at 40° C. for one hour.Polysaccharides which interfere with filtration have been degraded bythen. Better and quicker filterability on the vacuum rotational filterensues.

Example O

975 g of (water content ≦1%) and 20 g of fumed silica (Aerosil 380®product of Degussa AG, W.-Germany) are dispersed in a stirring vesselusing a toothed-disk agitator at a rotative speed of 15-18 m/sec for 15minutes. Then 5 g of a pectinase concentrate from Aspergillus oryzaewith an activity of 100 000 PGU/mg are added and the mixture isdispersed for another 10 minutes. (For definition and measurement of PGUc.f. Rohm-Analoysen-vorschrift PZV-30).

In this way a liquid, homogeneous, readily pourable pectinaseformulation with 5 000 PGU/mg is obtained, which after storing at 25° C.for 6 weeks shows neither creaming nor settling.

The activity after this period of time was found to be 97% of theoriginal activity.

Example P

1 000 l of freshly pressed apple juice are treated with 50 g of thepectinase formulation according to example O. This causes degradation ofpectin as well as of colloidal substances among the turbidifyingmaterial present. Those colloids would interfere with settling andfurther clarification via ultrafiltration or microfiltration. After 2hours of treatment, settling is carried out by adding bentonite.Subsequent filtration is performed easily and without any problem.

Example Q

Liquid enzyme composition for the soaking of hides and skins

In a vessel, 300 g of (1) a nonionic surfactant made of C₁₃ -oxoalcoholcondensed with 6 moles of ethylene oxide, (12) 682 g of a nonionicsurfactant made of C₁₃ -oxoalcohol and 9 mol of ethylene oxide, (3) 13 gof Aerosil 380® (fumed silica, product of Degussa AG, W.-Germany), and(4) 5 g of Borchigen STL® a surface active agent are dispersed with atoothed-disk agitator at a rotative speed of 15-18 m/s for 15 minutes.Then 27.5 g of an alkaline protease from Bacillus subtilis (activity 80000 LVE/g) and 10 g of a fungal protease from Aspergillus parasiticus(activity 220 000 LVE/g) are added in this order and are dispersed foranother 10 minutes. One obtains a liquid, homogenous enzyme preparationwhich can be easily transferred by pouring or pumping and which has notsettled or creamed after 4 weeks. The activity after this period of timeat 15° C. is 96% of the original one, which was 4 400 LVE/g.

Example R

Enzymatic soaking of presoaked cattle hide (percentage figures givenrefer to the weight of the skin materials treated (salt weight)

100 kg of presoaked cattle pelts

200% of water (26° C.)

0.25% of the enzyme composition of example Q

0.2% of sodium hydroxide solution (50% water)

After 5 hours of soaking in a vat, salted cattle pelts are thoroughlysoftened. Most of the grease and scud is removed and the skin is readyfor hair loosening.

Example S

Liquid bating preparation with deliming and defatting effect 400 g ofethylene carbonate, 300 g of methyl ethylketone and 264 of nonionicsurfactant (C₁₆ -C₁₈ fatty alcohol condensed with 9 moles ethyleneoxide) are mixed and warmed to 40° C. in a stirring vessel. Then 10 g offumed silica (Aerosil 380®) and 6 g of Borchigen STL® (see example Q)are added. The mixture is dispersed by means of toothed-disk agitator at15-18 m/sec for 15 minutes. The viscosity rises from 60 mPas to 1040mPas. Then 20 g of pancreatic enzyme concentrate (activity 40 000 LVE/g)are added and the mixture is dispersed for another 20 minutes. Oneobtains a liquid homogeneous enzyme formulation which is easily pouredand which will neither settle nor cream during 5 weeks of storing at 25°C. Activity after this period was found to be 760 LVE (Original activitywas 800 LVE/g).

Example T

Enzymatic bating and deliming of cattle pelts (percentages refer to theweight of pelt material)

10 kg of limed and washed cattle split pelts, 3-4 mm thick are treatedwith 30% of water at 35° C., 1% of the enzyme formulation according toexample S (800 LVE/g) and 2 g of ammonium sulfate.

After 70 minutes the pelts are thoroughly delimed and the scud is wellloosened. The final pH value of the liquid was fdund to be 7.8. Thenatural grease is widely removed from the skin and is emulsified in theliquor.

Example U

984 g of 1,2-propylene glycol (anhydrous) are dispersed with 10 g offumed silica (Aerosil 380®) and 5 g of a face active agent (BorchigenSTL®) for 10 minutes by means of a toothed-disk agitator at 15-18 m/s ina beaker. The viscosity of the liquid rises from 20 mPas to 310 mPas.Then 1 g of horseradish peroxidase (activity 1000 units/g; fordefinition and determination cf. F. W. J. Toele, Biochem. Biophys. Acta35, 543 [1959] are added and the mixture is dispersed for another 10minutes. A liquid, readily flowable enzyme formulation is obtained (280mPas) which after four weeks of storage at room temperature showedneither settling nor creaming. The activity was reduced by about 2%compared with the original enzyme activity.

Example V

Preparation of a dye solution (model reaction)

solution (a)

1 g of m-phenylene diamine are dissolved in 999 g of 0.1 molar phosphatebuffer (pH=7).

solution )b)

0.35 g of hydrogen peroxide (30%)

Enzymatic oxidation process

1 g of the peroxidase formulation according to Example U are dissolvedin 1000 g of solution (a). 5 g of solution (b) are added with stirring.Formation of a red azo dye is observed, whose colour has become quiteintense after some minutes.

What is claimed is:
 1. A liquid enzyme formulation comprising at leastone enzyme dispersed in a substantially anhydrous 5- or 6-memberedcyclic carbonate ester as a liquid carrier vehicle therefor togetherwith from 0.1 to 6 percent, by weight of said organic liquid, of afinely divided solid inroganic dispersing agent.
 2. A liquid enzymeformulation as in claim 1 comprising a protease.
 3. A liquid enzymeformulation as in claim 1 comprising an amylase.
 4. A liquid enzymeformulation as in claim 1 comprising a lipase.
 5. A liquid enzymeformulation as in claim 1 comprising a catalase.
 6. A liquid enzymeformulation as in claim 1 wherein said finely divided solid inorganicdispersing agent is selected from the group consiting of water insolublesalts of the alkaline earth metals, aluminum hydroxide, talc, clays andsilicon dioxide.